Abstract

Loss of heterozygosity of several specific genomic regions is frequently
observed in neuroblastoma tumors and cell lines, but homozygous
deletion (HD) is rare, and no neuroblastoma tumor suppressor gene
(TSG) has yet been identified. We performed a systematic search
for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma
cell lines. An initial search focused on a well-characterized consensus
region of hemizygous deletion at 1p36.3, which occurs in 35% of
primary neuroblastomas. Each cell line was screened with 162 1p36
markers, for a resolution of 13 kb within the consensus 1p36.3 deletion
region and 350 kb throughout the remainder of 1p36. No HDs were
detected. This approach was expanded to survey 21 known TSGs,
specifically targeting intragenic regions frequently inactivated in
other malignancies. HD was detected only at the
CDKN2A
(p16INK4a/p14ARF)
gene at 9p21 and was observed in 4 of 46 cell lines. The observed
region of HD included all exons of both CDKN2A and the
closely linked CDKN2B
(p15INK4b) gene for cell lines LA-N-6
and CHLA-174, all exons of CDKN2A but none of
CDKN2B for CHLA-179, and only 104 bp within
CDKN2A exon 2 for CHLA-101. All four deletions are
predicted to inactivate the coding regions of both
p16INK4a and
p14ARF. HD was observed in
corresponding primary tumor samples for CHLA-101 and CHLA-174 but was
not present in constitutional samples. These results suggest that for
neuroblastoma, large HDs do not occur within 1p36, most known TSGs are
not homozygously deleted, and biallelic inactivation of
CDKN2A may contribute to tumorigenicity in a subset of
cases.

INTRODUCTION

The biallelic inactivation of
TSGs
3
is considered crucial for the genesis and evolution of
neoplastic cells. Biallelic inactivation of a specific TSG can occur by
several mechanisms, including chromosomal deletion of both alleles.
Although uncommon, HD is occasionally observed in primary tumors and
cell lines from a variety of malignancies, and the region of biallelic
deletion is usually confined to a small genomic region surrounding a
target TSG. As HDs usually span relatively short genomic regions, the
detection and characterization of HD in various malignancies has been
instrumental in the identification of several TSGs, including
RB1, WT1, and CDKN2A(1,
2,
3)
.

Neuroblastoma is a common pediatric tumor of the peripheral nervous
system and is often incurable when diagnosed after 1 year of age.
Neuroblastoma displays remarkable clinical heterogeneity, ranging from
spontaneous regression and/or differentiation to rapidly progressive
and metastatic disease, and considerable genetic heterogeneity is also
apparent. Cytogenetic, molecular genetic, and functional analyses of
primary tumors and cell lines have identified a number of genomic
regions frequently exhibiting hemizygous deletion
(4)
.
Deletion of 1p36 correlates strongly with advanced disease and is the
most well-characterized region of deletion in neuroblastoma
(5,
6,
7)
. LOH studies have narrowed the region of consistent
overlapping deletion to 1p36.3, and this region is deleted in ∼35%
of primary neuroblastomas
(5, 8, 9)
. Several groups have
also hypothesized the existence of one or more additional TSGs located
elsewhere within 1p36
(10,
11,
12)
. Despite the
characterization of numerous candidates, no 1p36 TSG has yet been
identified, largely because of the paucity of tumors with localized
1p36 rearrangements
(4)
.

Besides 1p36, several additional regions of the genome are frequently
deleted in primary neuroblastomas, including 3p, 4p, 11q23, and 14q32
(4)
. However, no TSG consistently mutated or rearranged in
neuroblastoma tumors has yet been identified, and analyses of several
known TSGs, including TP53, RET,
CDKN2A, and MADH4, have detected few if any
mutations in these genes
(4)
. Furthermore, HD of known
tumor suppressor loci has been reported only rarely in neuroblastoma
(13,
14,
15,
16)
. The lack of evidence for genetic alterations in
the genes encoding the DNA damage sensor p53 and the
cyclin-dependent kinase inhibitory protein
p16INK4a is notable, as these two genes
are commonly disrupted in most malignancies
(17, 18)
.

In the present study, we investigated whether HD occurs with
significant frequency in neuroblastoma by systematically screening a
large panel of cell lines with markers mapping to 1p36 and also with
markers representing known TSGs. We report evidence for HD at
CDKN2A but not within distal 1p or at other known TSGs in
our cell line panel.

MATERIALS AND METHODS

Sample Collection and DNA Isolation.

A panel of 46 neuroblastoma cell lines were used for HD analysis.
Twenty-four cell lines have been previously described (Table 1)
⇓
. Cell lines CHLA-10, CHLA-101, CHLA-103, CHLA-108, CHLA-124, CHLA-132,
CHLA-136, CHLA-138, CHLA-140, CHLA-143, CHLA-152, CHLA-153, CHLA-171,
CHLA-174, CHLA-178, CHLA-179, CHLA-185, CHLA-52, CHLA-54, CHLA-60,
CHLA-95, and CHLA-98 were established as described from patients
treated with myeloablative chemoradiotherapy
(19)
. Cell
lines CHP-901 and CHP-902R were established at the Children’s Hospital
of Philadelphia from a bone marrow biopsy and a relapsed tumor mass,
respectively, and cultured in RPMI 1640 (Life Technologies, Inc.,
Gaithersburg, MD) with 10% fetal bovine serum, 25 μg/ml gentamicin,
and 1% oxaloacetate/pyruvate/bovine insulin-media supplement
(OPI; Life Technologies, Inc.). MYCN amplification
and 1p allelic loss determinations were performed as described
previously
(7)
. Frozen tumor and corresponding normal
marrow samples of patients from which cell lines CHLA-101, CHLA-174,
and CHLA-179 were derived were obtained from the Children’s Cancer
Group Neuroblastoma Biology Resource Laboratory at Children’s Hospital
Los Angeles. To obtain primary tumor DNA from the patient who gave rise
to the LA-N-6 cell line, cryopreserved marrow was thawed, washed to
remove DMSO in Iscove’s DMEM with 10% fetal bovine serum, and treated
with 10 units/ml of DNase for 1 h at 37°C in a 5%
CO2 incubator. The mononuclear cells were
separated using a Ficoll density centrifugation. Mononuclear cells were
then treated with magnetic immunobeads (one bead/total cells) and tumor
cells removed as described previously
(20)
. The resulting
mononuclear cells, which contained <0.1% tumor, were centrifuged to a
pellet, medium-aspirated, and flash-frozen for future DNA extraction.
The Jurkat T-cell line was kindly provided by S. Lessin (University of
Pennsylvania). DNA was isolated from cell line pellets or frozen
tissue using anion-exchange chromatography (Qiagen, Valencia, CA). DNA
from Centre d’Etude du Polymorphisme Humain reference family
member 1331-01 (Coriell Cell Repositories, Camden, NJ) was used as a
normal control.

DNA Markers.

Details of the DNA markers used to screen for HD of 1p36 and other TSGs
are listed in Tables 2
⇓
and 3
⇓
, respectively. Primers used for detailed mapping of HD around
CDKN2A are included in Table 3
⇓
. Primer sequences were
obtained from the Genome
Database
4
and from literature reports of specific TSG characterizations or were
generated from a representative sequence using Primer
3.
5
If possible, TSG primers targeted gene regions commonly disrupted in
other tumors. Markers for 1p36 were mapped using a distal 1p-specific
mapping
panel
6
as described previously. Markers mapping to 9p were ordered using map
information from the HUGO chromosome 9 integrated map (Genome
Database accession no. 6276683) and genomic sequence tracts from
GenBank.
7

RESULTS

HD Screen of 1p36.

Neuroblastoma cell lines were used to simplify HD detection, because
the absence of nonmalignant stromal cells allows implementation of a
high-throughput true/false assay system. A set of 46 genetically
distinct cell lines was selected for analysis (Table 1)
⇓
. Thirty-nine of
the 46 cell lines (85%) had evidence of a 1p hemizygous deletion.
Previously, we identified a smallest overlapping region of consistent
deletion (SRO) spanning ∼1 Mb within 1p36.3 by LOH analyses of
primary neuroblastomas
(5)
. This region has been further
characterized by the construction and analysis of a distal 1p-specific
mapping panel, which subdivides 1p35–p36 into 50 distinct genomic
intervals
(21)
. The 1p36.3 SRO is entirely contained
within mapping intervals 6–11 (Table 2
⇓
; Ref.
5
).
Seventy-six markers within the SRO, providing an average HD detection
resolution of approximately 13 kb, were used to survey the 46
neuroblastoma cell lines by PCR. Included in this set of markers were
primers representing each gene or expressed sequence tag cluster known
to map within or near the SRO, including the proposed candidate
neuroblastoma TSGs TP73, TNFRSF12, and
HKR3(22,
23,
24)
. To our knowledge, HD for 1p36
has never been reported; therefore, we created a HD control by pooling
DNA from 10 RH cell lines known to contain several human DNA fragments
but no fragments within 1p36. A product of the predicted size was
detected with all 76 markers for every neuroblastoma cell line but not
for the HD control or a negative control (no template; Fig. 1A⇓
).

Survey of HD at D1S47 (1p36.3) and
CDKN2A exon 2 (9p21). Cell lines were amplified with
primers for D1S47 (A) and
CDKN2A exon 2 (B) and resolved by
horizontal gel electrophoresis. A, at
D1S47, a product of the predicted size was detected in
every neuroblastoma cell line and in a normal human control, but not in
the HD control (RH) or a negative control (no template).
B, the CDKN2A primers did not amplify a
product in three neuroblastoma cell lines (CHLA-174, CHLA-179, and
LA-N-6). In addition, a CDKN2A product ∼100 bp shorter
is present only in cell line CHLA-101, which is consistent with DNA
sequencing results showing a 104-bp deletion in this sample.

Although all reported 1p36 deletions in neuroblastomas include allelic
loss for the 1p36.3 SRO, additional 1p36 suppressor loci both distal
and proximal to this region have been postulated and a number of
candidate TSGs have been proposed
(4)
. Therefore, a second
set of 86 1p36 markers was used to assess whether HD might occur
elsewhere within 1p36 (Table 3)
⇓
. Markers mapping within each of the 50
intervals defined by the distal 1p mapping panel were included to
assure that each portion of 1p36 was well represented. We also designed
markers for the proposed 1p36 TSGs (TNFRSF1B,
RIZ, PAX7, NBL1, TCEB3,
E2F2, C1ORF4, LAP18, and
ID3; Ref.
4
), as well as for twelve
markers surrounding a reported 1p36.2 translocation breakpoint in the
neuroblastoma cell line NGP
(9, 25)
. The 86 markers, which
provide an average 1p36 resolution of 350 kb, were used to survey the
46 neuroblastoma cell lines. A product of the predicted size was
visible for each marker in every neuroblastoma cell line and in a
normal control but not in a negative control.

HD Screen of Known TSGs.

Several known TSGs have been characterized for mutations in
neuroblastomas, but no abnormalities have been detected with
significant frequency. However, most of these analyses have included
only small cohorts of tumors and/or cell lines, and many identified
TSGs have not been investigated. Therefore, we extended our HD search
to include 21 known TSGs throughout the genome (Table 3)
⇓
. Intragenic
primers suitable for genomic PCR were designed for each TSG. Whenever
possible, primers were selected to span intragenic regions previously
demonstrated to be the most frequently deleted and/or mutated in
malignant cells. The 21 TSG markers were each used to survey the
neuroblastoma cell line panel and the Jurkat T-cell line, which is
homozygously deleted for CDKN2A(26)
. Twenty of
the 21 TSG markers yielded an amplification product in every cell line.
However, the CDKN2A primers, which span exon 2 of the gene,
did not amplify a product in three neuroblastoma cell lines (CHLA-174,
CHLA-179, and LA-N-6), nor in the Jurkat control, in repeated trials
using both ethidium staining and radioisotopic detection (Fig. 1B)
⇓
. In addition, a CDKN2A product ∼100 bp
shorter than the predicted length was consistently generated in cell
line CHLA-101 (Fig. 1B)
⇓
and was the only product generated
in this cell line.

Analysis of CDKN2A Deletions.

The CDKN2A HDs detected in the four neuroblastoma cell lines
were investigated further with 28 additional markers located within or
flanking CDKN2A to map the proximal and distal HD
boundaries. Included were markers spanning exons 1α, 1β, and 3 and
introns 1α, 1β, and 2 of CDKN2A; exons 1 and 2 of
CDKN2B; and several proximal and distal loci (Fig. 2)
⇓
. LA-N-6, CHLA-174, and the control Jurkat line demonstrated HD for the
entire CDKN2A gene including exon 1β, which encodes the 5′
portion of p14ARF, and both exons of
CDKN2B. CHLA-179 showed HD for all exons of
CKDN2A but not for CDKN2B. No additional
alterations were detected for CHLA-101. A survey of the remaining 42
neuroblastoma cell lines with the four CDKN2A and two
CDKN2B exon-specific markers detected products of the
expected size in all cases. The LA-N-6, CHLA-174, and CHLA-179 HDs were
confined to a region of 9p21 flanked by IFNA and
D9S171 (Fig. 2)
⇓
, and each deletion would be predicted to
completely abolish production of both the p14ARF
and p16INK4a protein products.

Extent of HD in four neuroblastoma cell lines.
Top, map of 9p21 region surrounding
CDKN2A. Shaded boxes indicate
CDKN2B (p15) and CDKN2A (p16) exons.
Markers used in the HD assay are depicted by solid vertical
ticks. The map is to scale except for the most 5′ and 3′
flanking markers and is oriented relative to the chromosome 9
centromere (cen) and 9p telomere (tel).
Below, the extent of HD in the four neuroblastoma cell
lines. Horizontal black bars indicate the presence of at
least one copy of the region.

The CDKN2A HDs were then confirmed by Southern analysis
(Fig. 3)
⇓
. Corresponding primary tumor and constitutional DNAs for LA-N-6,
CHLA-174, and CHLA-179 were analyzed in parallel to determine whether
HD occurred in vivo or only after cell-culture
establishment. A CDKN2A exon 2 probe hybridized to the
requisite 3.4-kb PstI fragment in constitutional DNA samples
from the three cell lines showing HD by PCR, as well as in primary
tumor DNA from CHLA-179. However, in agreement with the PCR results, no
hybridization was observed for the three cell lines with
CKDN2A HD. Furthermore, primary tumor DNA for CHLA-174 also
failed to hybridize to the CDKN2A probe, suggesting that HD
occurred in vivo in this tumor (no LA-N-6 tumor sample was
available for analysis). Hybridization with a control probe from a
different chromosome to the same filter yielded a band of identical
size and approximately the same intensity for each sample (not shown).

Southern analysis of CDKN2A. Genomic DNA
samples were digested with PstI and hybridized with a
CDKN2A exon 2 probe that recognizes a 3.5 kb
PstI fragment. HD is apparent in the cell lines
Jurkat (negative control), LA-N-6, CHLA-179, and
CHLA-174 and in a corresponding primary tumor sample for CHLA-174.
CHLA-101 exhibits a band shift that is the result of a 104-bp
deletion entirely within exon 2. CL, cell line DNA;
T, primary tumor DNA; N, normal
(nonmalignant tissue) DNA. No tumor sample was available for LA-N-6.

Southern analysis of cell line CHLA-101 confirmed the PCR results of a
truncated CDKN2A exon 2 (Fig. 3)
⇓
. Both techniques
demonstrated an ∼100-bp deletion within exon 2. DNA sequencing of the
truncated exon 2 PCR product showed a deletion of 104 bp (bp 246–349
and residues 69–103 of p16INK4a; and bp 390–493
and residues 83–118 of p14ARF) entirely
contained within exon 2. PCR analysis of primary tumor DNA for the
patient from which CHLA-101 was derived showed only the truncated exon
2 fragment, suggesting that the primary tumor contained an exon 2 HD
identical to the cell line (not shown). Sequencing of CDKN2A
exon 2 from the primary tumor DNA confirmed these results. No
corresponding constitutional DNA for CHLA-101 was available for
analysis.

The 104-bp deletion of CDKN2A exon 2 in CHLA-101 is
predicted to disrupt both p16INK4a and
p14ARF, which use alternate reading frames within
exon 2. For p16INK4a, the COOH-terminal 87
residues of the wild-type protein would be replaced with the 15
COOH-terminal residues of p14ARF because of a
frameshift from the p16INK4a reading frame to the
p14ARF reading frame. It is unlikely that such a
fusion protein, if expressed in CHLA-101, would be functional, as the
deleted region of p16INK4a contains the Cdk4/Cdk6
binding site where most loss-of-function point mutations localize
(27)
. For p14ARF, the final 50
residues would be replaced by 42 frameshifted residues.

DISCUSSION

Hemizygous deletion of distal 1p, first identified in 1977, is
frequently observed in advanced stage neuroblastomas
(4)
.
However, despite the identification of a 1 Mb SRO for LOH within 1p36.3
(5)
, no distal 1p TSG has yet been identified. To date,>
1000 primary tumors have been assessed for 1p allelic loss, but no
small deletions or HD have been detected
(4)
. Furthermore,
germline, tumor, or cell line-specific 1p translocation breakpoints are
rare and scattered throughout 1p
(4)
. The lack of
localized 1p rearrangements has led to alternate hypotheses for the
mechanism of 1p-mediated tumor suppression. Several groups have pursued
additional 1p36 TSG loci by: (a) mapping 1p36-specific
primary tumor or cell line rearrangements identified in closely
associated malignancies
(28)
; (b)
characterizing constitutional 1p36 rearrangements in patients
subsequently developing neuroblastoma
(9, 29)
; or
(c) identifying correlations between 1p deletion size and
the presence of MYCN amplification
(11, 30)
.
Moreover, although not directly supported by the present study,
mechanisms other than genetically mediated biallelic inactivation of a
TSG may be applicable, including haploinsufficiency of one or more
1p36 loci or imprinting as an epigenetic mechanism for
inactivation of the remaining TSG allele.

Our findings of no HD within 1p36 are consistent with the possibility
that disruption of only a single 1p36 homologue can facilitate
neuroblastoma tumorigenesis. Although the marker density we used to
survey HD within the 1p36.3 SRO was high (13 kb), very small regions of
HD may have gone undetected. In most cases, biallelic inactivation of a
TSG occurs by intragenic deletion or bp mutation of one allele, and
then by a second, larger deletion event in the remaining allele, as is
common with TP53(31)
. Because we surveyed only
cultured cell lines for HD at 1p36.3, it is formally possible that HD
occurs more frequently in primary tumors. However, this seems unlikely,
given that many of the cell lines in our study were derived from
advanced neuroblastomas passaged repeatedly in vitro, and
that most cell lines had hemizygous deletion of distal 1p.

With the exception of CDKN2A, HD was not detected for any of
the 21 TSGs surveyed. Our inclusion of positive and negative controls
for HD and the confirmation of CDKN2A HD by Southern
analysis for all four identified HD cases suggests that our PCR-based
HD assay is both sensitive and specific. It remains possible that HD of
one or more TSGs went undetected with the exon primers used for TSG
surveying. However, because we targeted the most commonly disrupted
gene region of each TSG, it is likely that the majority of HD events
would have been identified. HDs and/or inactivating mutations in
neuroblastoma are known to be rare or absent in the TP53,
DCC, MADH4, and RET tumor suppressor
genes
(4)
. Previously, four cases of HD have been reported
for neuroblastoma: HD of several exons of the NF1 gene at
17q11.2 in a primary tumor from a patient with neurofibromatosis type 1
(13)
and in a neuroblastoma cell line
(32)
;
HD of the CASP8 gene at 2q33 in one cell line
(16)
; and HD of CDKN2A in a single cell line
(see below; Ref.
14
). Whereas we did not detect HD of
NF1, the NF1 locus has not yet been well
characterized in neuroblastoma, although there appears to be no
increased incidence of neuroblastoma in patients with neurofibromatosis
or vice versa(32)
. The present study does not
address whether HD occurs at other chromosomal loci than those included
here. However, experiments using representational differential analysis
did not detect HD in several neuroblastoma cell
lines.
8
Furthermore, a lack of HD at a tumor suppressor locus does not preclude
inactivation of the gene by other genetic or epigenetic events, such as
gene mutation or methylation-mediated gene silencing.

The region of 9p21 is frequently deleted in a wide range of
malignancies
(33)
. Three loci in 9p21 have been implicated
as TSGs: CDKN2A/p16INK4a and
CDKN2A/p14ARF, which partially share a
coding exon but are encoded by two distinct reading frames; and the
highly homologous CDKN2B/p15INK4b
(Fig. 2
⇓
; Refs.
3, 34
). Substantial genetic evidence
suggests that disruption of both p16INK4a and
p14ARF, but not p15INK4b,
is crucial for tumor development
(18)
. Almost all
tumor-specific rearrangements of 9p21 alter exon 2, which is shared by
p16INK4a and p14ARF, and
only a few mutations affecting just one of the three implicated
proteins have been reported
(18, 35)
. Targeted deletion
experiments of the three loci in mice also suggest a causative role for
CDKN2A but not CDKN2B, as mice with germ-line
disruptions of CDKN2A are cancer-prone
(36)
.
p16INK4a acts as an inhibitor of the cell cycle
activators cdk4 and cdk6, which in turn inactivate the pRB tumor
suppressor protein, whereas p14ARF is thought to
derepress p53 by binding to and inactivating mdm2
(18)
.
Disruptions of the CDKN2A locus that concomitantly eliminate
functional p16INK4a and
p14ARF are thus believed to inactivate both the
p53 and Rb tumor suppression pathways.

Inactivation of both the Rb and p53 pathways are key events for
tumorigenicity in almost all neoplasms
(31, 37)
. Because
mutations and gene rearrangements of TP53 and RB1
are rare in neuroblastoma, these pathways might instead be disrupted by
other mechanisms. One possible alternative is a CDKN2A gene
rearrangement that simultaneously inactivates both
p16INK4a and p14ARF. The
four neuroblastoma HDs described in the present study all target
CDKN2A exon 2 and disrupt both the
p16INK4a and p14ARF coding
regions, consistent with the type of CDKN2A rearrangements
seen in other malignancies. The exon 2-specific 104-bp
CDKN2A deletion observed in cell line CHLA-101 supports this
hypothesis. Also, because the CHLA-101 and CHLA-179 HDs do not extend
to CDKN2B, it is unlikely that biallelic disruption of
CDKN2B plays a role in neuroblastoma tumorigenesis, although
monoallelic deletion of this gene could conceivably have an ancillary
effect. However, mutations or deletions limited to CDKN2B
have not been detected in neuroblastomas
(38)
.

Previous genetic analyses have reported evidence for infrequent
disruption of CDKN2A in neuroblastoma. Several LOH studies
cumulatively found 9p21 allelic loss in 29 of 131 (22%) primary
neuroblastomas
(14, 38,
39,
40,
41)
, with LOH most frequently
observed in tumors identified by mass screening of urinary
catecholamine metabolites. However, HD of CDKN2A has not
been found in primary tumors
(3, 14, 38, 39, 41,
42,
43)
,
although Dicicianni et al.(14)
reported HD for
an adriamycin-resistant subclone of the Be2C cell line that was not
observed in the parent cell line. Comparative genomic hybridization
studies of primary neuroblastomas have also reported only a low
frequency of 9p deletions
(4)
. Furthermore, only a single
CDKN2A mutation, a missense mutation at residue 52 of
p16INK4a in exon 2 has been identified out of 178
primary tumors and 28 cell lines screened
(14, 38, 39, 41,
42,
43)
. Notably, exon 1β (encoding
p14ARF) has not been included in any of the
mutation screens, but these results together suggest that a purely
genetic disruption of the CDKN2A locus is uncommon. Our
results agree with these findings in that only 4 of 46 (9%) cell lines
demonstrate gross biallelic inactivation. Nevertheless, our
identification of HD in two of three corresponding primary tumors
suggests that CDKN2A inactivation is a contributing in
vivo genetic event for a subset of neuroblastomas, rather than a
strictly in vitro phenomenon.

It is also conceivable that p16INK4a and/or
p14ARF are inactivated by alternative mechanisms.
A correlation between promoter hypermethylation and decreased
expression of the p16INK4a transcript, presumably
providing an epigenetic mechanism for CDKN2A suppression,
has been noted in other malignancies
(44,
45,
46)
. Three
studies of CDKN2A methylation in neuroblastoma cumulatively
found exon 1α methylation in 35% of tumors and cell lines, but no
correlations between methylation status and expression levels were
apparent
(14, 38, 41)
. The methylation status and
mutational analysis of CDKN2A exon 1β in neuroblastoma has
not been reported. The expression pattern of CDKN2A in
primary tumors is varied and exhibits no obvious correlations with
other parameters
(38, 41, 47)
. Likewise, analyses of other
genes in the CDKN2A pathway have found few abnormalities in
neuroblastoma
(14, 38, 42, 47,
48,
49)
.

Mutation of the TP53 gene is very rare in primary
neuroblastomas, and deletion or LOH of 17p is uncommon
(4)
. There is, however, evidence that inactivation of the
p53 pathway is important in some neuroblastomas. Amplification of the
p53-inhibitory MDM2 locus has been identified in several
neuroblastoma cell lines and in a single tumor
(50,
51,
52)
.
Furthermore, immunohistochemistry studies suggest that p53 is
sequestered in the cytoplasm in neuroblastomas, which has been
postulated as a posttranslational mechanism for functional repression
of p53
(53, 54)
. The CDKN2A deletions observed
in the present study, which would be predicted to affect p53 function
through p14ARF, are consistent with alterations
in p53 signaling playing a role in the tumorigenesis of some
neuroblastomas. More detailed analysis of p14ARF
expression and function in neuroblastoma will be needed to fully
understand the contribution that this protein plays in neuroblastoma.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.